Neisseria gonorrhoeae, the causative agent for the sexually transmitted infection gonorrhea, was responsible for an estimated 850,000 infections in the U.S. in 2012 and 106 million cases worldwide. Untreated or untreatable infections can lead to infertility, pelvic inflammatory disease (PID) in females, gonococcal arthritis in both sexes, and an increased risk of both contracting and transmitting HIV. Over the past few years, the steady and inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonorrhea infections. Most alarmingly, verified treatment failures against the extended-spectrum cephalosporins (ESCs), cefixime and ceftriaxone, have now been reported, prompting the Centers for Disease Control to recommend dual antibiotic therapy to replace the monotherapy that has been effective for ~80 years. Further increases in antibiotic resistance appear inevitable. This precarious position endangers public health and demands a better understanding of antibiotic resistance at the molecular level to enable countermeasures. A sentinel event in the development of cephalosporin resistance (CephR) in N. gonorrhoeae was the emergence of the first pan-resistant strain, called H041. Comparison of the known resistance mechanisms in H041 with those of cephalosporin-intermediate resistant strains (CephI) shows that alterations in the penA gene are responsible for the elevation to CephR. penA encodes penicillin-binding protein (PBP) 2, an essential transpeptidase (TPase) that functions in peptidoglycan synthesis. This renewal application proposes an investigation of the structural mechanisms underpinning cephalosporin resistance in N. gonorrhoeae due to mutations in PBP2. Crystal structures of PBP2, including complexes with ?-lactams, will be solved to elucidate how key mutations present in penA from H041 lower reactivity of the enzyme with ESCs. We will also conduct biochemical and structural investigations of the interactions between PBP2 variants and PG substrates, including boronic acid probes of the transition state, to determine how mutations selectively discriminate against ?-lactams without abrogating TPase activity. Finally, to examine our hypothesis that protein dynamics plays a critical role in antibiotic resistance, we will use NMR approaches to investigate whether mutations in PBP2 conferring cephalosporin resistance enhance or reduce the population of an alternative conformational state, and whether this state represents a productive or inhibitory conformation for the acylation reaction with both ?-lactams and PG substrate. By revealing the molecular mechanisms of how PBPs overcome the lethal action of ?-lactams, these investigations will enable new strategies for the development of replacement anti-gonococcal agents.